Cervical cancer screening is undergoing a major transformation with the adoption of testing for the presence of oncogenic human papilloma virus (HPV) types (1, 2). Persistent HPV infection of the cervical epithelium is a rare event because most infections are usually cleared without treatment (3). Yet a small percentage of HPV infections are associated to progression from low-grade squamous intraepithelial lesions (LSIL) to cervical intraepithelial neoplasia grade 3 (CIN3) lesions (4). In fact, persistent infection with at least one of the 13 carcinogenic types from the alphapapillomavirus genus (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68) has been linked to cervical cancer (5). Close to 30% of CIN3 lesions progress to cervical cancer, but there are no agreed upon clinical tests of progression to cervical cancer (6, 7).
The new cervical cancer screening guidelines in the United States recommend HPV co-testing with Pap among women 30 years and older (8). However, clinical management for HPV-positive/Pap-negative women is not firmly established (9, 10). HPV has recently been shown to be a better indicator of cervical cancer risk than the Pap test (11). But even when co-testing with Pap and HPV has higher sensitivity and specificity than each separate test, together they cannot predict who will progress to cervical carcinoma (12, 13).
This led to the search of molecular markers of risk to progression to cervical cancer. HPV DNA methylation, like other DNA viruses, is correlated to progression to cancer (14). This seminal finding led to subsequent studies suggesting that detection of methylated HPV DNA may distinguish women with cervical intraepithelial neoplasia grade 2-3 (CIN2+) from women with an oncogenic HPV type infection who show no evidence of CIN2+ (15-19). In parallel, other groups examined the association between host DNA methylation and cervical cancer (20-23). Recent studies, have reported a positive association between CIN2+ and methylation of CpG sites in host and viral DNA isolated from liquid cytology samples (19, 20, 24).
Recently published reports have also explored the use of urine-based high risk HPV testing, as an alternative approach to liquid cytology for cervical cancer screening, in an attempt to identify less invasive cervical cancer screening technologies (25-27). Most of the studies have failed to attain clinical usefulness as they are limited by poor sensitivity, inappropriate protocols for DNA extraction from circulating DNA in urine, small sample size, and the limit of detection of the HPV assays utilized (28-30). Most of the studies also fail to recognize the importance of using DNA isolation methods optimized to enrich for fragmented circulating cell-free DNA (ccfDNA) that crosses the kidneys and can be obtained in urine (31).
There is growing evidence that circulating short human, viral, and bacterial DNA fragments from dying cells throughout the body, approximately 150-250 bases long on average, pass through the renal barrier and appear in urine as Transrenal DNA (TrDNA)(32) or ccfDNA. ccfDNA has been proposed as a tool for non-invasive prenatal monitoring, infectious disease monitoring, and tumor response monitoring (33-35). Recently, a capillary electrophoresis ccfDNA test that targets the E1 region of the HPV genome for the detection of high risk HPV demonstrated high sensitivity and modest specificity for urine-based detection of cervical pre-cancerous lesions (36).
Urine samples tested by the ccfDNA HPV capillary electrophoresis test had high concordance with corresponding cervical and urine samples tested by the widely used Linear Array HPV Genotyping Test (LA-HPV)(37). However, the ccfDNA capillary electrophoresis HPV test is not quantitative, can only detect the presence or absence of high risk HPV and, similar to previously published urine based HPV tests, has limited specificity. None of the high-risk HPV urine-based reports use sequencing based approaches to quantify multiple HPV types, nor include methylated markers in their workflow to improve the sensitivity and specificity of the ccfDNA test.